The long-range goal of this investigation is the complete analysis of the energetics of protein-protein and protein-effector interactions in the allosteric enzyme aspartate transcarbamylase from E. coli. Previous work has dealt with the energetics of binding of substrate analogs to the native enzyme and catalytic subunit (Knier & Allewell, 1978; Hofmann et al., 1979; Allewell et al., 1979) and the binding of nucleoside triphosphates to the native enzyme over a restricted range of conditions (Allewell et al., 1975). The emphasis during the next grant period will be on the energetics of protein-protein interactions, and the changes in the subunit interaction energies produced by various perturbations of the structure. Our goals are (a) to define the thermodynamic basis of assembly, and (b) to provide rigorous thermodynamic criteria against which models of the allosteric mechanism can be tested. In the first phase of the work, the energetics of the interactions between (a) C and R subunits in the native enzme, (b) individual c chains in the C subunit, and (c) individual r chains in the R subunit will be examined, as a function of pH, temperature and ionic strength. In addition, hydrogen exchange will be used to evaluate the magnitude of the structural changes produced by protein-protein interactions, to identify regions perturbed by the interaction, and to partition the overall energy change among the perturbed regions. The second phase of the work will involve using the same approaches to analyze the changes in the subunit interaction energies which result from various perturbations of the structure (i.e., ligand binding, chemical modification or mutaton, and elimination or substitution of the metal ion) as a function of environmental conditions. These measurements will allow the question of the allosteric mechanism to be addressed and may permit critical interactions at the C:R intrface to be identified. While various experimental tecniques will be used as needed, the emphasis will be on microcalorimetery, hydrogen exchange, potentiometry, and radioactive binding studies.